As the terminal residue of membrane-associated glycoconjugates in vertebrates and higher invertebrates, sialic acids are important parts of the cellular apparatus devoted to the detection and integration of environmental stimuli. Several pathogenic microorganisms are known to incorporate sialic acids into their surface structures, thereby mimicking an abundant molecular component displayed by host cells, and providing a mechanism to evade immune response (Vimr, E. & Lichtensteiger, C., Trends Microbiol., 10, 254-257 (2002)).
Sialic acids are a family of carboxylated monosaccharides that possess a common backbone structure of 9 carbon atoms. The structural diversity in the family arises as a result of various modifications to the biochemical precursor, and most naturally abundant member, 5-N-acetyl-neuraminic acid (NeuAc) (Schauer, R., Glycobiology. 1, 449-452 (1991); Varki, A., Glycobiology, 2, 25-40 (1992)). One commonly observed modification is O-acetylation at one or more of the hydroxyl groups at positions 4, 7, 8 or 9. This process, which is catalyzed enzymatically by sialate-O-acetyltransferases (SOATs), changes the binding and recognition characteristics of the underlying molecule, and as a result, diversifies the interaction potential for a given sialoglycan.
O-acetylation of sialic acids has been implicated in a growing number of physiological and pathological processes. In developing animals, ganglioside O-acetylation is associated with tissue growth and differentiation (Constantine-Paton, M. et al., Nature, 324, 459-462 (1986); Shi, W. X. et al. J. Biol. Chem., 271, 31517-31525. (1996)). This process is of clinical importance because different modification patterns have been observed in human cell lines that have undergone malignant transformation (Cheresh, D. A. et al., Science, 225, 844-846. (1984); Hutchins, J. T. et al. Cancer Res., 48, 483-489 (1988)). Therefore O-acetylated gangliosides could serve as targets for directed cancer therapies. O-acetylation has contrasting effects on the process of viral attachment to membrane-bound sialoglycans; this is an obligatory modification for the association of some enteric and respiratory viruses, but inhibits binding by others (Herrler, G. et al. EMBO J., 4, 1503-1506 (1985); Smits, S. L. et al., J. Biol. Chem., 280, 6933-6941 (2005)). As a final example, the sialylated polysaccharide capsules of group B Streptococcus (Lewis, A. L. et al., Proc. Natl. Acad. Sci. USA, 101, 11123-11128 (2004)), Escherichia coli K1 (11) (Orskov, F. et al., J. Exp. Med., 149, 669-685 (1979)) and Neisseria meningitidis serogroup C, W-135 and Y (Bhattacharjee, A. et al., Can. J. Biochem., 54, 1-8 (1976)) have been shown to be O-acetylated in some cases, which results in altered immunogenic properties.
The mucosal pathogen Campylobacter jejuni is a leading cause of diarrheal disease and of food-borne gastroenteritis worldwide (Nachamkin, I. et al., Clin. Microbiol., Rev. 11, 555-567 (1998)). This organism exhibits a highly variable array of cell-surface glycans that are associated with virulence (Gilbert, M. et al., J. Biol. Chem., 277, 327-337 (2002); Szymanski, C. M. et al., J. Biol. Chem., 278, 24509-24520 (2003)). In several strains of C. jejuni, the glycan component of the lipo-oligosaccharide (LOS) is sialylated, and structurally similar to gangliosides (Aspinall, G. O. et al., Eur. J. Biochem., 213, 1017-1027 (1993); Aspinall, G. O. et al., Eur. J. Biochem., 213, 1029-1037 (1993); Aspinall, G. O. et al., Biochemistry 33, 241-249 (1994); St Michael, F. et al., Eur. J. Biochem., 269, 5119-5136 (2002)). There is an accumulating body of evidence to suggest that the LOS from these strains may be responsible for generating antibodies that are cross-reactive with host epitopes found in abundance in nervous tissue, triggering an auto-immune response, which gives rise to Guillain-Barré syndrome (Godschalk, P. C. et al., J. Clin. Invest., 114, 1659-1665 (2004); Yuki, N. et al., Proc. Natl. Acad. Sci. USA, 101, 11404-11409 (2004)).
As yet, there is no conclusive biochemical data to confirm the presence of O-acetylated NeuAc in the LOS of C. jejuni strains. This, we believe, is a result of the unique challenge inherent in the characterization of the glycan component of LOS; conventional spectroscopic techniques require the prior removal of the fatty acyl components. Unfortunately, chemical treatments used to accomplish this task have the undesired consequence of cleaving NeuAc residues, and/or saponification of O-acetyl modifications. NeuAc has been found to be incorporated into the LOS of Haemophilus influenzae (Hood, D. W. et al., Mol. Microbiol., 33, 679-692 (1999)), Neisseria spp. (Smith, H., Microb. Pathog., 19, 365-377 (1995)), and C. jejuni (Aspinall, G. O. et al., Biochemistry, 33, 241-249 (1994)), however, there have been no reports of O-acetylated species. Without evidence of the O-acetylated species, there has been a related failure to identify bacterial enzymes that synthesize such compounds. The present invention meets this and other needs.